Oxidative-Induced Angiogenesis Is Modulated by Small Extracellular Vesicle miR-302a-3p Cargo in Retinal Pigment Epithelium Cells

Extracellular vesicles are released from cells under diverse conditions. Widely studied in cancer, they are associated with different diseases playing major roles. Recent reports indicate that oxidative damage promotes the release of small extracellular vesicle (sEVs) from the retinal pigment epithelium (RPE), with an angiogenic outcome and changes in micro-RNA (miRNA) levels. The aim of this study was to determine the role of the miRNA miR-302a-3p, included within RPE-released sEVs, as an angiogenic regulator in cultures of endothelial cells (HUVEC). ARPE-19 cell cultures, treated with H2O2 to cause an oxidative insult, were transfected with a miR-302a-3p mimic. Later, sEVs from the medium were isolated and added into HUVEC or ARPE-19 cultures. sEVs from ARPE-19 cells under oxidative damage presented a decrease of miR-302a-3p levels and exhibited proangiogenic properties. In contrast, sEVs from miR-302a-3p-mimic transfected cells resulted in control angiogenic levels. The results herein indicate that miR-302a-3p contained in sEVs can modify VEGFA mRNA expression levels as part of its antiangiogenic features.

Lately, miRNA-non-coding, short (around 22 nucleotides), single-stranded RNA molecules have also been highlighted as potential therapeutic tools and diagnostic markers [17][18][19][20]. miRNAs are able to interact with mRNA strains, inhibiting them, thus reducing protein expression. They are found inside the cell as well as the extracellular medium, including plasma. Circulating miRNAs might travel free in blood plasma or, as mentioned The arising retinal pigment epithelium (ARPE-19) human cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). ARPE-19 cells were cultured in Dulbecco's modified Eagle's medium/F12 (Invitrogen, Carlsbad, CA, USA), as previously described [24]. Cells were used from 11 to 30 passages. Cells were cultured to 80-90% confluence at a seeding density of 1 × 10 6 cells/cm 2 . After 48 h of seeding, cells were treated with 600 µM H 2 O 2 (Scharlau, Barcelona, Spain) and/or 4 mM N-acetylcysteine (NAC; Sigma-Aldrich, St. Louis, MI, USA) for 24 h grown in media supplemented with 1% of exosome-depleted fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA). Cell media and cells were collected and preserved for future experiments.

Vasculogenesis Assay
Vasculogenesis was performed in Matrigel (Becton Dickinson, Franklin Lakes, NJ, USA) as previously described [36]. Briefly, HUVEC were seeded 8 × 10 4 cells per well in a 96 well plate and then treated with ARPE-19 cell media for 5 h. The different conditions were determined by the conditioned medium used: control media, sEVs from control media, sEVs from mimic-miR-302a-3p transfected-control media, sEVs from 600 µM H 2 O 2 media, sEVs from mimic-miR-302a-3p transfected 600 µM H 2 O 2 media and 4 mM NAC media. Matrigel was allowed to solidify overnight at 37 • C. Pictures were taken with an Olympus CKX41 inverted microscope (Olympus, Tokyo, Japan) and recorded by an Olympus DP74 digital camera (Olympus, Tokyo, Japan). Angiogenesis Analyzer for ImageJ was used to analyze the total tube length of the taken images.

sEVs Isolation
sEVs isolation was performed using the Total Exosome Isolation Reagent from cell culture media (Thermo Fisher Scientific, Waltham, MA, USA). The 2 mL of cell media was centrifuged at 2000× g for 30 min to remove cells and debris. The supernatant containing the cell-free culture media was mixed with 1 mL of Total Exosome Isolation reagent and incubated overnight at 4 • C. After incubation, the sample was centrifuged at 10,000× g for 1 h at 4 • C. The pellet was resuspended in cell culture media and stored at −80 • C for further analysis.

sEV Characterization
sEV identity was confirmed by electronic microscopy and a nanoparticle tracking system, NanoSight NS300, as described previously [4].

Analysis of Pathways and miRNA Target Genes
In silico analysis of miR-302a-3p targets were performed using TargetScanHuman (https://www.targetscan.org/vert_80/; accessed on 4 September 2020) using Tarbase. The pathway analysis in which the detected miRNAs were involved was performed using DIANA TOOLS mirPath v.3 algorithm (http://snf-515788.vm.okeanos.grnet.gr/; accessed on 7 September 2020) [37]. Moreover, the analysis of the pathways regulated by the putative targets of the miRNAs was performed with the FUNRICH software using the gen ontology database. With STRING (https://string-db.org/; accessed on 20 September 2020), the networking of the targets was performed.

Transfection of miR-302a-3p Mimic
ARPE-19 cells were transfected with miR-302a-3p mirVana ® miRNA mimic (Thermo Fisher Scientific, Waltham, MA, USA), and mirVana™ miRNA mimic negative control #1 (Thermo Fisher Scientific, Waltham, MA, USA) as a negative control. The procedure of transfection of miRNA mimic and control was described previously [24]. After 48 h of transfection, ARPE-19 cells and cell culture media were collected for following analysis.

miRNA Expression Analysis
qRT-PCR was performed to assess sEV miRNA expression levels. sEV RNA was retrotranscribed using a TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Bedford, MA, USA) using the specific TaqMan RT primer for miR-302a-3p. qPCR was performed using the TaqMan™ microRNA primer for miR-302a-3p (Thermo Fisher Scientific, Waltham, MA, USA) with TaqMan Gene Expression Master Mix (Applied Biosystems, Bedford, MA, USA) and RT-PCR Roche 234 LighterCycler 480, as described by Oltra et al. [24]. RNU6B snoRNA was used as normalizer. Relative expression was calculated as 2 −∆∆Ct .

miR-302a-3p Targets and Related Pathways
Networks and biological pathways regulated by the predicted targets were entered into the FUNRICH software (version 3.

miR-302a-3p Targets and Related Pathways
Networks and biological pathways regulated by the predicted targets were entered into the FUNRICH software (version 3.1.3) to perform the functional analysis. The functional enrichment analysis of miR-302a-3p showed 607 biological processes regulated by at least one of the 1019 mRNA target of miR-302a-3p. Among the top 20 related pathways, 1.3% of genes were related to GTPase activity, dentate gyrus development by 0.7%, and DNA damage response by 3% (Figure 3). 1.3% of genes were related to GTPase activity, dentate gyrus development by 0.7%, and DNA damage response by 3% ( Figure 3). About 38 pathways related to miR-302a-3p mRNA targets are related to blood vessel formation. Using STRING, angiogenic and vasculogenic related processes were selected, and 213 mRNA predicted targets were found, which encode proteins involved in the regulation of angiogenic processes ( Figure 4A). Among the most repeated mRNA targets re-−Log10 (p-value) p-value Figure 3. miR-302a-3p protein target-related pathways. Top 20 biological processes regulated by miR-302a-3p predicted targets, using FUNRICH and the Gene Ontology database. About 38 pathways related to miR-302a-3p mRNA targets are related to blood vessel formation. Using STRING, angiogenic and vasculogenic related processes were selected, and 213 mRNA predicted targets were found, which encode proteins involved in the regulation of angiogenic processes ( Figure 4A). Among the most repeated mRNA targets related to vasculogenic processes are VEGFA, EGFR, and AKT1. In fact, in silico analysis showed that VEGFA, EGFR, and AKT1 mRNAs are predicted targets for miR-302a-3p ( Figure 4B).

VEGFA mRNA and Angiogenesis Were Regulated by H2O2 and SEVs
To assess the effect of sEVs on VEGFA mRNA expression, sEVs from both contro and H2O2-treated ARPE-19 cells were isolated and cultured with naïve ARPE-19 cells. W previously reported that VEGFA mRNA was significantly overexpressed in ARPE-19 cel after H2O2 exposure [24]. Similarly, ARPE-19 cells cultured with H2O2-induced sEVs re sulted in significantly increased VEGFA mRNA levels compared to control and contro induced sEVs ( Figure 5E). In order to demonstrate the role of sEVs in angiogenesis, HU VEC cultured on standard cell culture medium or with sEVs from control ARPE-19 cel did not result in significant levels of angiogenesis ( Figure 5A,B,D). However, cell cultur media with H2O2-induced sEVs significantly increased angiogenesis in HUVEC cells (Fig  ure 5C,D).

VEGFA mRNA and Angiogenesis Were Regulated by H 2 O 2 and SEVs
To assess the effect of sEVs on VEGFA mRNA expression, sEVs from both control and H 2 O 2 -treated ARPE-19 cells were isolated and cultured with naïve ARPE-19 cells. We previously reported that VEGFA mRNA was significantly overexpressed in ARPE-19 cells after H 2 O 2 exposure [24]. Similarly, ARPE-19 cells cultured with H 2 O 2 -induced sEVs resulted in significantly increased VEGFA mRNA levels compared to control and control-induced sEVs ( Figure 5E). In order to demonstrate the role of sEVs in angiogenesis, HUVEC cultured on standard cell culture medium or with sEVs from control ARPE-19 cells did not result in significant levels of angiogenesis ( Figure 5A,B,D). However, cell culture media with H 2 O 2 -induced sEVs significantly increased angiogenesis in HUVEC cells ( Figure 5C,D).

miR-302a-3p sEV Cargo Regulated Oxidative-Induced Angiogenesis
HUVEC vasculogenic assay was performed to demonstrate the angiogenic effect of miR-302a-3p. HUVEC were grown with sEVs released from ARPE-19 cells under different conditions. Control-released sEVs keep the vascular processes at levels equals to fresh cell culture medium ( Figure 6A,B,I). Moreover, sEVs released after miR-302a-3p mimic addition resulted in a significant vascular decrease ( Figure 6D,I). As expected, H2O2-induced sEVs significantly increased angiogenic changes ( Figure 6F,J). However, these changes were reduced by adding miR-302a-3p mimic (with H2O2 treatment, see the Materials and Methods section) ( Figure 6G,J). All the aforementioned changes were normalized by NAC addition, indicating its oxidative nature (Figure 6C,E,G,I,J). Values are expressed as mean ± SEM (n = 3; Matrigel assay and n = 5; mRNA expression analysis). The p-value was obtained by ANOVA; * p < 0.05 and ** p < 0.01.

miR-302a-3p sEV Cargo Regulated Oxidative-Induced Angiogenesis
HUVEC vasculogenic assay was performed to demonstrate the angiogenic effect of miR-302a-3p. HUVEC were grown with sEVs released from ARPE-19 cells under different conditions. Control-released sEVs keep the vascular processes at levels equals to fresh cell culture medium ( Figure 6A,B,I). Moreover, sEVs released after miR-302a-3p mimic addition resulted in a significant vascular decrease ( Figure 6D,I). As expected, H 2 O 2 -induced sEVs significantly increased angiogenic changes ( Figure 6F,J). However, these changes were reduced by adding miR-302a-3p mimic (with H 2 O 2 treatment, see the Materials and Methods section) ( Figure 6G,J). All the aforementioned changes were normalized by NAC addition, indicating its oxidative nature (Figure 6C,E,G,I,J).

VEGFA mRNA Was Overexpressed by H2O2 and Modulated by miR-302a-3p
In silico analysis indicated miR-302a-3p as a VEGFA target. ARPE-19 VEGFA mRNA expression was determined by qRT-PCR (Figure 7). VEGFA mRNA was significantly overexpressed by both H2O2 sEVs and H2O2 exposure compared to control sEVs and the control. miR-302a-3p mimic exposure significantly decreased the H2O2-induced VEGFA mRNA expression under the control mRNA expression levels. The addition of NAC significantly reduced H2O2-induced VEGFA mRNA expression to control levels.

VEGFA mRNA Was Overexpressed by H 2 O 2 and Modulated by miR-302a-3p
In silico analysis indicated miR-302a-3p as a VEGFA target. ARPE-19 VEGFA mRNA expression was determined by qRT-PCR (Figure 7). VEGFA mRNA was significantly overexpressed by both H 2 O 2 sEVs and H 2 O 2 exposure compared to control sEVs and the control. miR-302a-3p mimic exposure significantly decreased the H 2 O 2 -induced VEGFA mRNA expression under the control mRNA expression levels. The addition of NAC significantly reduced H 2 O 2 -induced VEGFA mRNA expression to control levels.
Finally, in order to demonstrate that miR-302a-3p mimic exposure is capable of affecting sEV cargo, we transfected ARPE-19 cells with miR-302a-3p mimic (see the Materials and Methods section), and sEVs were analyzed by qRT-PCR. Our findings strongly indicated that miR-302a-3p mimic transfection resulted in a significantly high miR-302a-3p expression levels on sEVs (Figure 8). This result supports the possibility of a direct targeting of miR-302a-3p on VEGF mRNA.

Discussion
The functional meaning of sEV release is currently gaining relevance on different physiopathological processes. It has been recently reported how oxidative-induced sEVs released from human RPE present Drussen-related proteins as part of the cargo, and this could be related to the pathological processes undergone in AMD patients [23]. Additionally, under hypoxia A431 cells release more than 50% of cytosolic proteins related to angiogenesis as part of the sEVs cargo [38]. We report herein how H2O2-induced sEVs can modulate angiogenesis via miR-302a-3p-VEGFA mRNA modulation.
One of the first noteworthy aspects is that H2O2-induced sEVs are capable of transmitting this "oxidative" condition. H2O2-induced sEVs increased ROS and decreased ARPE-19 cell viability. This fact matches with other studies that showed that the oxidative insult modulates the number of sEVs released and the nature of their cargo [1,4,[39][40][41] After seeing NAC results, one can argue that ROS or indirect pro-oxidant molecules are included as sEVs cargo. NAC was administered to ARPE-19 treated with H2O2-induced sEVs cultures, resulting in ROS level reduction. In addition, this process was accompanied by significantly decreased miR-302a-3p inside the sEVs [4]. Recently, we demonstrated how H2O2 stimulate angiogenesis processes [24]. Here, we demonstrate how the H2O2induced sEVs can promote angiogenesis similarly to H2O2 exposure.
In order to demonstrate the role of miR-302a-3p in angiogenesis, we transfected ARPE-19 cells with a miR-302a-3p mimic on the different experimental conditions. The Values are expressed as mean ± SEM (n = 3). The p-value was calculated by t-test, and statistically significant differences were ** p < 0.01.

Discussion
The functional meaning of sEV release is currently gaining relevance on different physiopathological processes. It has been recently reported how oxidative-induced sEVs released from human RPE present Drussen-related proteins as part of the cargo, and this could be related to the pathological processes undergone in AMD patients [23]. Additionally, under hypoxia A431 cells release more than 50% of cytosolic proteins related to angiogenesis as part of the sEVs cargo [38]. We report herein how H 2 O 2 -induced sEVs can modulate angiogenesis via miR-302a-3p-VEGFA mRNA modulation.
One of the first noteworthy aspects is that H 2 O 2 -induced sEVs are capable of transmitting this "oxidative" condition. H 2 O 2 -induced sEVs increased ROS and decreased ARPE-19 cell viability. This fact matches with other studies that showed that the oxidative insult modulates the number of sEVs released and the nature of their cargo [1,4,[39][40][41]. After seeing NAC results, one can argue that ROS or indirect pro-oxidant molecules are included as sEVs cargo. NAC was administered to ARPE-19 treated with H 2 O 2 -induced sEVs cultures, resulting in ROS level reduction. In addition, this process was accompanied by significantly decreased miR-302a-3p inside the sEVs [4]. Recently, we demonstrated how H 2 O 2 stimulate angiogenesis processes [24]. Here, we demonstrate how the H 2 O 2 -induced sEVs can promote angiogenesis similarly to H 2 O 2 exposure.
In order to demonstrate the role of miR-302a-3p in angiogenesis, we transfected ARPE-19 cells with a miR-302a-3p mimic on the different experimental conditions. The results indicate a marked negative regulation of miR-302a-3p on VEGFA mRNA expression levels/angiogenesis. H 2 O 2 -induced sEVs transfected with miR-302a-3p mimic resulted in reduced level of angiogenesis/VEGFA mRNA expression. This finding again fits with the suggestion of a direct miR-302a-3p/VEGFA mRNA binding as a mechanism of action. Interestingly, sEVs from H 2 O 2 -treated ARPE-19 cells significantly increase angiogenesis and VEGFA mRNA expression compared to control-treated cells. This agrees with previous data from our lab indicating that oxidative stress significantly decreases miR-302a-3p levels in sEVs, compared to control [4], thus providing support to the anti-angiogenic role of miR-302a-3p. Furthermore, it is well characterized that oxidative stress can promote the angiogenic drive [48][49][50]. VEGFA is upregulated by ROS through the PI3K/Akt pathway [51]. The addition of the antioxidant NAC to stressed condition normalized the increased VEGF mRNA/angiogenesis levels, suggesting it as an oxidative-dependent event.
In conclusion, oxidative stress promotes sEV release with pro-angionenic properties. The angiogenic effect is inhibited by miR-302a-3p and promoted by ROS. The mechanism of action seems to be related to the topological homology between VEGFA mRNA and miR-302a-3p. This opens a new strategy against oxidative-induced angiogenesis by promoting miR-302a-3p as a VEGFA mRNA repressor.